Rapid replication is characteristic of virulence in certain bacteria, viruses and malignancies, but no chemistry common to rapid replication in different organisms has been described previously. This patent application discloses a new class of protein structures related to rapid replication. A new family of conserved small proteins related to rapid replication, named Replikins, which are used to predict and control rapid replication in multiple organisms and diseases and to induce rapid replication in plant and animal life.
We constructed an algorithm search for Replikins. In applying the algorithm invented herein not only was the function of the epitope revealed—rapid replication, but an entire family of homologues whose function is related to rapid replication was discovered, which we named Replikins.
The algorithm is based on the following: 1) Evidence that the immune system looks to parts rather than a whole protein in recognition. Protein chains are first hydrolyzed by the immune system into smaller pieces, frequently six (6) to ten (10) amino acids long, as part of the immune systems' process of recognition of foreign structures against which it may mount an immune defense. By way of example, the immune system recognizes the presence of disease by chopping up proteins of the disease agent into smaller peptide sequences and reading them. This principle is used as a basis for the algorithm with which to search for homologues of the malignin cancer epitope, once the structure of the epitope was known; 2) The specific structure of the malignin epitope, in which two of the three lysines (K's) are eight residues apart is in accordance with the apparent ‘rules’ used by the immune system for recognition referred to above (6-10 amino acids long); 3) The fact that the malignin cancer epitope was shown to be a very strong antigen, that is—a generator of a strong immune response; that there are three lysines (K's) in the 10-mer peptide glioma Replikin and that K's are known to bind frequently to DNA and RNA as potential anchors for the entry of viruses; and 4) One histidine (H) is included in the sequence of the malignin epitope, between the two K's which are eight (8) residues apart, suggesting a connection to the metals of redox systems which are required to provide the energy for replication.
Engineered enzymes and catalytic antibodies, possessing tailored binding pockets with appropriately positioned functional groups have been successful in catalyzing a number of chemical transformations, sometimes with impressive efficiencies. Just as two or more separate proteins with specific and quite different functions are now often recognized to be synthesized together by organisms, and then separately cleaved to ‘go about their separate functions’, so the Replikin structure is a unique protein with a unique function that appears to be recognized separately by the immune system and may be now rationally engineered—e.g. synthesized to produce a functional unit.
From a proteomic point of view, this template based on the newly determined glioma peptide sequence has led to the discovery of a wide class of proteins with related conserved structures and a particular function, in this case replication. Examples of the increase in Replikin concentration with virulence of a disease appear in diseases including, influenza, HIV, cancer and tomato leaf curl virus. This class of structures is related to the phenomenon of rapid replication in organisms as diverse as yeast, algae, plants, the gemini curl leaf tomato virus, HIV and cancer.
In addition to detecting the presence of Replikins in rapidly replicating organisms, we found that 1) Replikin concentration (number of Replikins per 100 amino acids) and 2) Replikin compositions in specific functional states dependant on rapid replication, provide the basis for the finding that Replikins are related quantitatively as well as qualitatively to the rate of replication of the organism in which they reside. Examples of these functional proofs include the relationship found between rapid replication and virulence in glioblastoma cells, between Replikins in influenza virus and the prediction of influenza pandemics and epidemics, and the relationship between Replikin concentration and rapid replication in HIV.
The first functional basis for Replikins' role in rapid replication was found in the properties of the glioma Replikin, a 10 KD peptide called Malignin in brain glioblastoma multiforme (glioma)—a 250 KD cell protein. Antimalignin antibody increased in concentration in serum (AMAS), measured by an early stage diagnostic test for cancer now used for most or all cell types. Malignin was so named because in tissue culture the expression of this peptide and its concentration per milligram membrane protein extractable increased with increased rate of cell division per unit time. Not only is there an increase in the amount of malignin in proportion to the cell number increase but the amount of malignin is enriched, that is—increased ten fold whereas the cell number increased only five fold.
The structure of malignin protein was determined through hydrolysis and mass spectrometry which revealed what proved to be a novel 16 mer peptide sequence. We searched for the 16 mer peptide sequence which we have named a Glioma Replikin protein in databases for the healthy human genome and found that it was not present in these databases.
As such, the fixed requirement algorithm was used to search in other organisms for the Glioma Replikin protein or homologues thereof. Over 4,000 protein sequences in the “Pub Med” database were searched and homologues were found in viruses and plant forms specifically associated with rapid replication. Homologues of such Replikin proteins occurred frequently in proteins called ‘replicating proteins’ by their investigators.
Homologues of the Replikin sequence were found in all tumor viruses (that is viruses that cause cancer), and in ‘replicating proteins’ of algae, plants, fungi, viruses and bacteria.
That malignin is enriched ten-fold compared to the five-fold increase in cell number and membrane protein concentration in rapid replication of glioma cells suggests an integral relationship of the Replikins to replication. When the glioma replikin was synthesized in vitro and administered as a synthetic vaccine to rabbits, abundant antimalignin antibody was produced—establishing rigourously the antigenic basis of the antimalignin antibody in serum (AMAS) test, and providing the first potential synthetic cancer vaccine and the prototype for Replikin vaccines in other organisms.
The demonstration of the relationship of the Replikins to replication and the natural immune response to cancer Replikins (overriding cell type) based upon the shared specificity of cancer Replikins, permits passive augmentation of immunity with antimalignin antibody and active augmentation with synthetic Replikin vaccines.
A study of 8,090 serum specimens from cancer patients and controls has demonstrated that the concentration of antimalignin antibody increases with age in healthy individuals, as the incidence of cancer in the population increases, and increases further two to three-fold in early malignancy, regardless of cell type. In vitro this antibody is cytotoxic to cancer cells at picograms (femtomoles) per cancer cell, and in vivo the concentration of antimalignin antibody relates quantitatively to the survival of cancer patients. As shown in glioma cells, the stage in cancer at which cells only have been transformed to the immortal malignant state but remain quiescent or dormant, now can be distinguished from the more active life-threatening replicating state which is characterized by the increased concentration of Replikins. In addition, clues to the viral pathogenesis of cancer may be found in the fact that glioma glycoprotein 10B has a 50% reduction in carbohydrate residues when compared to the normal 10B. This reduction is associated with virus entry in other instances and so may be evidence of the attachment of virus for the delivery of virus Replikins to the 10B of glial cells as a step in the transformation to the malignant state.
The sharing of immunological specificity by diverse members of the class, as demonstrated with antimalignin antibody for the glioma and related cancer Replikins, suggests that B cells and their product antibodies may recognize Replikins by means of a similar recognition ‘language’. With the discovery of the Replikins, this shared immunological specificity may explain what was previously difficult to understand: why the antimalignin antibody is elevated in all cancers, and is cytotoxic to cancer cells and related to survival of cancer patients in most or all cell types. Thus antimalignin antibody is produced against cancer Replikins, which share immunological specificity and which are related to the phenomenon of rapid replication, not to cell type.
A second functional basis for the Replikins' role in rapid replication is the study of data from the past 100 years on influenza virus hemagglutinin protein sequences and epidemiology of influenza epidemics and pandemics. To date, only serological hemagglutinin and antibody classification, but no strain-specific conserved peptide sequences have previously been described in influenza, and no changes in concentration and composition of any strain-specific peptide sequences have been described previously which correlate with epidemiologically documented epidemics or rapid replication.
A four to ten-fold increase in the concentration of strain-specific influenza Replikins in one of each of the four major strains, influenza B, (A)H1N1, (A)H2N2 and (A)H3N2 was found, and that such increase of Replikin concentration was related to influenza epidemics caused specifically by each strain from 1902 to 2001. These increases in concentration were then shown to be due to the reappearance of at least one specific Replikin composition from 1 to up to 64 years after its disappearance, plus the emergence of new strain-specific Replikin compositions. Previously, no strain-specific chemical structures were known with which to predict which strains would predominate in coming influenza seasons, nor to devise annual mixtures of whole-virus strains for vaccines. The recent sharp increase in H3N2 Replikin concentration (1997 to 2000), the largest in H3N2's history, and the reappearance of specific Replikin compositions which were last seen in the high mortality H3N2 pandemic of 1968 and in the two high mortality epidemics of 1975 and 1977, but were absent for 20-25 years, together may be a warning of coming epidemics.
Synthetic Replikins are new vaccines. This high degree of conservation of Replikin structures observed whereby the identical structure can persist for 100 years, or reappear after an absence of from one to 64 years reappears indicates that what was previously thought to be change in virulence due to random substitution of amino acids in influenza proteins is more likely to be change due to an organized process of conservation of Replikins. In fact, if random substitutions of each amino acid occurred, the chance against an average length influenza Replikin sequence being conserved for one year (let alone 100) is calculated to be in the order of 2 to the 27th power to 1.
The significant conservation of Replikins is not unique to influenza virus is also present in foot and mouth disease virus type O and in HIV, as well as in wheat.
A third functional basis for Replikins' role in rapid replication is the increase in Replikin concentration shown to be related to rapid replication in HIV. The Replikin concentration in the slow-growing low-titre strain of HIV (NS1, “Bru”), prevalent in early stage infection, was found to be one-sixth of the Replikin concentration in the rapidly-growing high-titre strain of HIV (SI, “Lai”), prevalent in late stage HIV infection.
Other examples are given of the relation of Replikins to rapid replication. For example, in tomato curl leaf gemini virus, which devastates tomato crops, the first 161 amino acids of the ‘replicating protein’, which have been shown to bind to DNA, contain five Replikins.
In malaria, legendary for rapid replication, trypanosomes are released from the liver in tens of thousands from one trypanosome. Multiple, novel, almost ‘flamboyant’ Replikin structures with concentrations of up to 36 overlapping Replikins per 100 amino acids are found therein.
The increase in Replikin concentration in influenza epidemics is functionally comparable to the glioma Replikin's increase in concentration during rapid replication of malignant glioma cells and comparable to rapid replication in HIV and in a diverse range of other organisms. Replikins thus are associated with and appear to be part of the structural bases of rapid replication in different organisms.
Replikin concentration and composition therefore provide new methods to detect and to control the process of replication, which is central to the survival and dominance of each biological population. The discovery of these new proteins related to rapid replication provides new opportunities 1) for detection of pathogens by qualitative and quantitative determinations of Replikins, 2) for the control of a broad range of diseases in which rapid replication is a key factor by targeting native Replikins and by using synthetic Replikins as vaccines, and 3) for the use of Replikins to foster growth of algal and plant foods.
There is a significant number of diseases and pathogens which have proved difficult to detect and treat and for which there is no effective vaccine. Thus, for each disorder there is a need for developing a target that will provide effective methods of detecting, treating or preventing these diseases and pathogens.